Spores of Ascosphaera apis contained in wax foundation

can infect honeybee brood

J.M. Flores
a,
*
, M. Spivak
b
, I. Gutierrez
a
a
Centro Andaluz de Apicultura Ecologica, Universidad de Cordoba,
Campus Universitario de Rabanales, 14071 Cordoba, Spain
b
Department of Entomology, University of Minnesota, 219 Hodson Hall, St Paul, MN 55108, USA
Received 24 November 2004; received in revised form 11 March 2005; accepted 30 March 2005
Abstract
Chalkbrood disease in honeybees (Apis mellifera L.) is caused by an infection with Ascosphaera apis. Disease expression
requires the consumption of fungal spores and a predisposing condition in the susceptible brood. A. apis spores within sheets of
wax foundation could be a source of inoculum leading to chalkbrood, but it is also possible that these spores remain conned in
the wax and do not contribute to disease. We have resolved this topic by chilling susceptible brood within wax combs built on
contaminated foundation (using treatments of spores from 1 mummy and spores from 10 mummies) versus uncontaminated
foundation. We found signicantly higher levels of chalkbrood in brood exposed to the higher dosage. Our results demonstrate
that foundation wax contaminated with spores of A. apis spores may be a source of chalkbrood in honeybee colonies.
# 2005 Elsevier B.V. All rights reserved.
Keywords: Honeybee; Apis mellifera; Ascosphaera apis; Chalkbrood; Spores; Disease; Wax foundation
1. Introduction
Chalkbrood disease in honeybees (Apis mellifera
L.) is caused by an infection with Ascosphaera apis
(Olive and Spiltoir) which affects the developing
brood. Larvae ingest the fungal spores when feeding,
permitting the disease to develop in the stretched
larvae after sealing. The stretched larvae are killed and
later dry, leaving a mummied cadaver reminiscent of
a small piece of chalk, which become dark if fruiting
bodies of the fungi are formed (sporulated mummies).
This disease requires a predisposing condition in the
susceptible brood for it to develop (reviewed by Heath,
1982). Larvae in the fth stage, prior to and some
hours after sealing, are most susceptible to the disease
(Bailey, 1967; Puerta et al., 1994; Flores et al., 1996).
To preserve the wholesome and natural character-
istics of honey and other beekeeping products,
alternative methods must be developed to control
chalkbrood, to avoid the use of fungicides, which risk
contaminating hive products. An important precau-
tionary measure that beekeepers can take is to avoid
transferring wax combs between colonies that contain
chalkbrood infection and spores, because the occur-
www.elsevier.com/locate/vetmic
Veterinary Microbiology 108 (2005) 141144
* Corresponding author. Tel.: +34 957 218698;
fax: +34 957 218698.
E-mail address: ba1sej@uco.es (J.M. Flores).
0378-1135/$ see front matter # 2005 Elsevier B.V. All rights reserved.
doi:10.1016/j.vetmic.2005.03.005
rence of the disease is proportional to the quantity of
circulating spores (Puerta et al., 1990).
Any material with A. apis spores that has contact
with bees can result in disease transmission (reviewed
by Heath, 1982; Gilliam, 1990). It has been speculated
that spores within the wax foundation (the wax base
used in beekeeping on which bees draw out wax cells
for brood development or honey and pollen storage)
are a source of infection. However, this conjecture
could not be conrmed until techniques were
developed to allow the experimental expression of
clinical symptoms in a controlled way while main-
taining the natural conditions of the beehives as far as
possible (Puerta et al., 1994; Flores et al., 1996). Now,
by chilling susceptible brood to predispose them to
infection (Puerta et al., 1994; Flores et al., 1996), we
have been able to demonstrate the role of these spores
in the expression of chalkbrood disease.
2. Materials and methods
Sporulated mummies were ground to obtain spores
of chalkbrood. The number of spores per mummy was
quantied using a Neubauer counting chamber and a
compound microscope (400). We obtained a mean
value of 80,000 spores/mummy.
Sheets of foundation were manufactured starting
from commercial wax. The wax was melted in a
microwave. We added the spores of the fungus when
temperatures were below 80 8C. The sheets of
foundation were made with 140 g of wax per sheet,
pouring the wax in a manual wax foundation mould
(Thomas
1
). The sheets cooled quickly and conse-
quently, the time which the spores were maintained at
this temperature was brief, minimizing the possibility
of any exposure to this temperature affecting spore
survival (Anderson et al., 1997).
Treatments consisted of foundation without spores
(control) (A), foundation contaminated with spores
from one mummy (approximately 571 spores/g wax)
(B), and foundation contaminated with spores from 10
mummies (approximately 5710 spores/g wax) (C).
The experiment was performed in the Andalusian
Center for Organic Beekeeping (Cordoba, Spain)
during spring and early summer (2002), coinciding
with the beekeeping season. Trials were carried out in
three healthy Langstroth hives. Each colony was used
to test all three treatments. Sheets of foundation were
introduced progressively in the beehives. First, a sheet
of control foundation was introduced into each colony.
When the wax cells were drawn, and there were fth
instar bee brood in the cells, we introduced foundation
with spores from one mummy and later, in the same
way, we introduced foundation with spores from 10
mummies. The process was repeated three times per
colony (therefore each treatment was repeated three
times per colony). We began the research in spring,
when there were large numbers of eggs and young
larvae in the combs. The interval between introducing
two successive treatments was 1214 days. Also, we
alternated the treatments in three consecutives
repetitions in each colony (18: control, 28: one
mummy, 38: 10 mummies; 48: control, 58: one
mummy, 98: 10 mummies), and we repeated the
experiment in three colonies; thus, we reduced the
effect of climatic conditions.
The risk of becoming infected with chalkbrood
disease was evaluated in newly capped brood (worker
brood sealed within a period of 14 h) for each
treatment comb. Unsealed fth instar larvae (Rembold
et al., 1980) were marked on plastic sheets. The brood
combs were subsequently returned to the beehives and
again removed after 14 h. Portions of combs with
newly sealed brood were cut, removed and maintained
in incubators at 25 8C and 60% relative humidity to
chill the brood for 5 days. After that the cells were
opened and the percentage of mummied larvae was
determined (Flores et al., 1996).
In each test foundation we only used the rst
generation of bee brood, because cocoons from these
larvae could conne the spores contained in the wax,
being inaccessible to the following larval generations.
When the combs were lled with honey or pollen, they
were removed and replaced by a new sheet of
foundation with the same treatment.
The data obtained were evaluated statistically using
descriptive parameters (mean percentage of chalk-
brood S.E.); analysis of variance (one-way ANOVA)
and post hoc tests (Tukey honest signicant difference
(HSD) test, P < 0.05). SPSS 8.0 for Windows.
3. Results and discussion
Results are shown in Table 1.
J.M. Flores et al. / Veterinary Microbiology 108 (2005) 141144 142
The spores of A. apis contained within sheets of
wax foundation could be the source of the chalkbrood
for healthy bee colonies, but it is possible too that
these spores remain conned in the wax, without being
an important risk for the transmission of the disease.
We have resolved this topic by chilling susceptible
brood within wax combs built on contaminated
foundation.
Statistical analysis showed that the high dose
treatment had a signicant effect on the incidence of
chalkbrood symptoms (one-way ANOVA among
treatments, F = 6.746, d.f. = 2, P = 0.002). In parti-
cular, Tukey HSD tests revealed signicant differ-
ences were found between wax foundation without
spores (treatment A controls) and wax foundation
contaminated with the spores from 10 mummies
(treatment C). We did not observe signicant
differences between treatment B (wax foundation
contaminated with the spores of one mummy) and the
controls or treatment C.
Nevertheless, 2.04% of the brood showed chalk-
brood symptoms in the controls. It is possible that the
commercial wax used for manufacture of foundation
was contaminated. However, the results given in
Table 1 show that colonies 2 and 3 had a statistically
higher percentage of chalkbrood symptoms compared
to colony 1 (one-way ANOVA among colonies within
treatment A. F = 6.329, d.f. = 2, P = 0.003). We
hypothesize that although the commercial wax was
free of spores, the expression of chalkbrood was due to
the circulating spores in each colony.
On the other hand, we did not nd signicant
differences among colonies within treatments B (one-
way ANOVA, F = 0.969, d.f. = 2, P = 0.386) or C
(one-way ANOVA, F = 0.434, d.f. = 2, P = 0.650).
Incontrast, whenwe analyzedthe differences among
treatments within each colony, we found signicant
differences among treatments for colony 1 (one-way
ANOVA, F = 7.386, d.f. = 2, P = 0.002), but not for
colony 2 (F = 1.066, d.f. = 2, P = 0.351) or colony 3
(F = 1.355, d.f. = 2, P = 0.266). However, we did
observe higher disease levels for spore-contaminated
combs in all colonies. Our interpretation is that colony 1
had less background contamination, and the infected
waxfoundation signicantlyincreased the riskof larvae
contracting chalkbrood. In contrast, colonies 2 and 3
had higher background quantities of spores, so any
effect of the spores contained within the foundation was
masked by the circulating spores.
We conclude that the spores of chalkbrood
contained in foundation combs are a potential risk
for the dispersion of the disease, and research should
be conducted on preventives measure for the
elimination of these spores.
Acknowledgements
Funds were provided by the INIA (project API99-
007) supported by the EC and the Spanish Govern-
ment (CE 1221/97).
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Table 1
Number of investigated cells (susceptible larvae) and mean percentage of chalkbrood S.E. in colonies receiving three different treatments:
control foundation combs without spores (A), foundation combs contaminated with the spores from one mummy of chalkbrood (B) and
foundation combs contaminated with the spores from 10 mummies (C)
Treatment A control Treatment B Treatment C One-way ANOVA
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Number of
investigated cells
Chalkbrood
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Number of
investigated cells
Chalkbrood
(%)
Number of
investigated cells
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(%)
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Colony 3 3178 2.85 0.49 3347 3.52 0.51 3018 4.02 0.51 0.266
Total 7538 2.04 0.26 a 9606 2.94 0.32 ab 8524 3.84 0.43 b 0.002
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